Growth characteristics vary widely from one microorganism to another. For example, it has been estimated that relatively-rapidly growing Mycobacteria require approximately one week to demonstrate growth, whereas relatively more slowly-growing tuberculosis agents, such as M. tuberculosis, M. bovis and M. avium, which are also known to appear in AIDS patients, require at least eight to ten weeks of incubation under conventional conditions before growth is detectable by standard methods. As a result, methods for the rapid detection and accurate measurement of the cell growth of various microorganisms are useful for a variety of purposes, including monitoring yields in the production of microorganisms in industrial fermentation processes and the early detection of pathogenic microorganisms.
Growing microorganisms in liquid culture is a well-known technique. See, for instance, Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York); Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York); and Gerhardt et al. (eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, D.C.). When microorganisms are grown in a liquid culture medium, an accurate measure of the rate of oxygen depletion in the liquid culture medium can be used to determine, the presence of viable organisms in the culture following inoculation, as well as the rate of growth of that organism in the culture (see, e.g., U.S. Pat. No. 6,165,741).
Growth in liquid culture, however, is less useful for rapid identification of a slow-growing microorganism, particularly if there is a mixture of microorganisms and one seeks to identify individual clonal colonies. The longer a microorganism is cultured, the greater the risk of contamination, usually by a fast-growing bacteria, yeast or fungus. Fast-growing microorganisms tend to out-compete slow-growing microorganisms and overgrow the culture, obscuring the slow-growing microorganisms. Furthermore, in such a mixture of microorganisms, one can no longer identify and select individual cells for isolated growth.
Plating microorganisms onto a layer of agar or other growth media has long been used to separate individual microorganisms and permit isolating clonal growth of individual cells. Such techniques are well known to the skilled artisan. See, for instance, Sambrook et al., supra, 1989; Ausubel et al., supra, 1997; Gerhardt et al., supra, 1994). Plating slow-growing microorganisms, however, still requires long incubation periods to detect growth by conventional means, increasing the risk of contamination over time and increased handling.
Several methods are known for the detection of cell growth, such as U.S. Pat. No. 5,523,214, which describes a method for visually demonstrating the cell growth in broths or gels of microorganisms. Microorganisms include, for example, fungi, yeasts and bacteria, including Mycobacteria, non-fermenters, cocci, bacilli, coccobacilli, enterobacteria and the like, obtained from urine specimens, matter from wounds and abscesses, blood, sputum, etc. However, the detection method of the '214 patent utilize a redox indicator in the medium, meaning that the method is not commercially practical because the amount of redox indicator that is required to demonstrate growth of the microorganism may also be toxic to the cells, and/or such methods require an inordinate amount of care to avoid toxicity and prevent false negative results. Furthermore, like all other prior art methods, when the '214 method is used to detect the growth of slow-growing cells in culture, the cells require several weeks in culture before microbial cell growth is demonstrated. To date, with the exception of the inventor's own work, none of the available detection methods provide rapid and reliable detection of cell growth in culture in a matter of only a day or two.
Based upon the principle that oxygen quenches phosphorescence in an aqueous liquid growth medium, U.S. Pat. No. 6,165,741 provides one method for detecting growth or metabolism of microorganisms in culture. In a liquid culture medium containing a dissolved oxygen-quenchable phosphorescent compound, as the microbial sample grows, oxygen is consumed, and the oxygen quenching of the phosphorescent compound decreases. In other words, phosphorescence, indicative of growth or metabolism of the microorganisms, increases in direct ratio with cell growth and is quantitatively detectable at measurable levels in the culture medium. However, the typical volume of liquid culture media used to grow the cells in standard plates or vessels in the '741 patent requires the use of substantial volumes of reagent and marker materials, and while more rapid than other methods, several days are needed to provide reliable readings using this method. Moreover, because liquid culture techniques are used, the '741 patent does not permit the rapid identification and isolation of individual clonal colonies.
Hooijmans et al. ((1990) Appl. Microbiol. Biotechnol. 33:611-618) teach measurement of time-dependent oxygen concentration gradients of E. coli immobilized in gel beads or a cylindrical tube gel. Oxygen is measured using an oxygen microsensor comprising a flow chamber, a micromanipulator and a stereomicroscope, then the measured data is captured by a computer. However, the complexity of this method and the requirement of using specialized equipment, including an oxygen microsensor, makes it unsuitable for rapid growth detection purposes.
Lähdesmädki et al. ((1989) Analyst 125:1889-1895) disclose immobilizing cells in an agarose matrix, which is sandwiched between a physical support and the surface of an electrochemical pH sensor (Cytosensor Microphysiometer). A fluorescent technique is used to monitor change in pH of the growing microorganisms. In addition, the investigator discloses the use of disposable samples of cells grown on microcarrier beads labeled with a fluorescent pH indicator for use with the electrochemical pH sensor. However, this method suffers from the same drawbacks as the other prior art methods. Although a fluorescent indicator is used, because the cells are not immobilized, the disclosed methods cannot provide a means for isolating an individual cell or colony from the culture media.
Jeffrey et al. (U.S. Pat. No. 6,777,226) disclose a sensor device for detecting microorganisms. The sensor device discloses a multilayer construct, having a matrix layer to immobilize the microorganisms, and a sensor layer comprising a fluorescent indicator, which is used to monitor growth of the microorganisms. However, in the '226 patent, the sensor layer is a separate entity from the immobilization matrix layer.
Thus, until the present invention there has remained an unmet need in the art for a method of monitoring and rapidly detecting cell growth using a simple system that does not require complex microsensors, layering or risk of toxicity to the cells that could produce false negatives, while at the same time allowing for the collection of the individual colonies for further growth, manipulation and evaluation. Preferably, such detection methods would produce reliable results in a day or less, and would permit rapid and sensitive detection of cell growth down to statistical limits in terms of number of organisms per sample. Furthermore, such a method should be optimally adapted to an automated format.